capping cofactor Dcp1p, the decapping activator Dhh1p, Pat1p, Edc3p, the Lsm1p7p complex, and Xrn1p. These factors form macroscopic aggregates that appear as punctate cytoplasmic foci termed mRNA processing bodies . The sequences of the mammalian homologs Dcp1a and Dcp1b are similar to yeast Dcp1p in their N-terminal regions. The N terminus of mammalian Dcp1a contains an enabled vasodilatorstimulated protein homology 1/Wiskott-Aldrich syndrome protein 1 Phosphorylation of Dcp1a by ERK homology 1 functional domain, which is a proteininteracting domain that is conserved among eukaryotic Dcp1 proteins. Although mammalian Dcp1 homologs are conserved at the N terminus, the long C terminus of the mammalian Dcp1 homologs shows no similarity to yeast Dcp1p. Yeast Dcp1p can physically interact with Dcp2p, and recombinant Dcp1p can enhance the decapping activity of yeast Dcp2p. A similar interaction has not been observed in human cells, but Hedls/Ge1/Edc4 is believed to be the essential factor 17149874 responsible for the assembly of the decapping complex. Recent kinetic and structural studies of yeast decapping complexes have provided clues to how Dcp2p activity is regulated by Dcp1p. Mammalian Dcp1a has little homology with yeast Dcp1p, and little is known about the PBTZ 169 details of the mammalian decapping complex assembly or the regulation of decapping activity. In this study, we observed that Dcp1a was phosphorylated in response to differentiation signals in 3T3-L1 cells. Using mass spectrometry analysis combined with in vivo and in vitro phosphorylation assays and site-directed mutagenesis, we identified Ser315 and Ser319 as being phosphorylated by the ERK signaling pathway. We also demonstrated that the physical interaction of Dcp1a with Dcp2 was enhanced by phosphorylation at Ser315 and Ser319. The possible functional effect of Dcp1a phosphorylation in regulating ARE-containing mRNA degradation was investigated in the early differentiation of 3T3-L1 cells. Results Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of 3T3-L1 10712926 preadipocytes We previously demonstrated that the mRNA expression of some immediate early genes such as tristetraprolin and MAPK phosphatase-1 is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes. We thus examined whether mRNA decapping machinery plays a role in the expression of these IEGs. 3T3-L1 preadipocytes were induced to differentiate by incubation with an adipogenic cocktail comprised of methylisobutylmethylxanthine, dexamethasone, bovine insulin, and fetal bovine serum . Interestingly, the expression profile analysis of decapping protein Dcp1a revealed that endogenous mouse Dcp1a consisted of multiple bands on western blots, with the lower bands being more prevalent when cells were not induced. As the preadipocytes started to differentiate, the upper bands of Dcp1a became predominant 1 to 4 h after induction. After 8 h of induction, the predominant forms of Dcp1a became the lower bands again. We then examined whether the shifted Dcp1a bands 
included phosphorylated Dcp1a by treating cell lysates from the differentiation time course with calf intestinal phosphatase . CIP treatment resulted in a complete loss of the upper bands, suggesting that Dcp1a was phosphorylated and that the upper bands represented phosphorylated forms of Dcp1a. To identify the kinase responsible for Dcp1a phosphorylation, each inducer of differentiation was examined separately. Two days after
